Longitudinal Determination of Co-receptor Usage Using Ultra-deep Pyrosequencing
Charlotte Hedskog*1, J Jernberg1, J Albert1,2, and M Mild1
1Karolinska Inst, Stockholm, Sweden and 2Karolinska Univ Hosp, Stockholm, Sweden
Background: The entry of HIV-1 into target cells is dependent on the binding of the envelope glycoprotein to its receptor CD4 and a co-receptor, either CCR5 or CXCR4. Early in infection, the viral population generally uses CCR5 (R5-virus) and in approximately 50% of infected individuals the co-receptor usage switches or broadens to include CXCR4 (X4-virus) during disease progression. Such co-receptor switch is associated with an accelerated loss of CD4+ T cells and faster progression to AIDS.
Methods: We have used ultra-deep pyrosequencing (UDPS) to sequence the V3 loop of the viral envelope to investigate whether X4-virus are present as a minority species already during primary infection. Three to 6 samples from 3 patients who experience a co-receptor switch have been analyzed longitudinally from primary infection until after the switch. The UDPS reads have been filtered to remove errors introduced during PCR and sequencing. Viral tropism was determined using MT-2 assay and co-receptor use was predicted with bioinformatic algorithms (PSSMx4/r5 and Geno2Pheno[coreceptor]).
Results: Between 480 and 20,893 reads were generated from each sample. In one individual low-abundant CXCR4-using viruses were found during primary infection. However phylogenetic analysis showed that these viruses were not genetically similar to the X4 population detected after co-receptor switch. Furthermore, phylogenetic analysis showed that co-existing R5 and X4 populations present after the switch either clustered together or were found in separate clusters separated in the tree.
Conclusions: UDPS analysis has shown that for these 3 patients, X4-virus that grows after co-receptor switch is not present in primary infection and that the X4 population most probably develops from the R5 population of each patient during the course of infection.